Determining structural dynamics of membrane proteins in their native environment: focus on bacterial antibiotic resistance

细胞健康是由其生物大分子的结构、运动和相互作用决定的。能够在天然细胞环境中询问生物大分子的行为将使我们能够更好地了解这些分子如何决定细胞的行为和功能。蛋白质是一类重要的生物大分子，其执行广泛的细胞过程，如酶催化、细胞信号传导和支架以及DNA复制。它们由定义为多肽的线性氨基酸链组成，其序列由编码它们的遗传序列决定。蛋白质的一个重要子集是整合膜蛋白，其存在于细胞膜内并占细胞蛋白质的约30%。细胞膜是主要由蛋白质和脂质组成的动态结构，其作用是将细胞区室化，为细胞及其细胞器的外部环境提供屏障。整合膜蛋白由其疏水多肽段的含量定义，所述疏水多肽段使得其结构的部分能够嵌入细胞膜内或与细胞膜缔合。它们负责各种动态细胞过程，如感觉、细胞调节和细胞间粘附。膜蛋白的功能能力和它们的表达水平将在很大程度上决定离子组成，从而决定给定细胞类型的代谢水平，使它们对所有生命都是必不可少的，以及关键的药物靶点。我的主要目的是确定膜蛋白直接在其天然细胞膜环境，包括活细胞内的结构动态信息。理解蛋白质的结构动力学是很重要的，因为它们的波动经常代表对蛋白质功能至关重要的运动和状态。要做到这一点，我将制定一般策略，使膜蛋白的结构和动力学在复杂的环境中被破译先进的结构质谱方法。结构质谱使用多肽及其肽构建块的高分辨率质量信息来推断蛋白质分子的结构特性-它们的形状，相互作用和运动。使用诸如氢/氘交换质谱（其测量蛋白质骨架酰胺氢交换氘的程度和速率）的技术，可以传递关于蛋白质相互作用、配体结合和结构动力学的全局和局部信息。在这里，我提出了发展化学生物学和先进的质谱策略膜蛋白结构的调查在不同的天然membranesignification.One关键领域，其中完整的膜蛋白是重要的是在抗菌药物耐药性的发展。对抗抗生素耐药性是一项关键的社会挑战，如果不加以解决，有可能成为全球健康危机。在细菌细胞系中，对结构上不相关的化学物质的多重耐药性的发展与多药外排膜蛋白转运蛋白的功能相关，其排出广泛的有毒物质并导致抗生素的抑制作用降低。我的研究将集中在开发多药外排膜蛋白系统的背景下，这是已知的细菌抗生素耐药性中发挥重要作用的上述方法。这将使我们能够前所未有地深入了解这些系统的结构、动力学和功能，特别是药物和脂质相互作用的影响以及临床相关突变。更一般地说，对细胞内生物大分子进行结构洞察的能力将是我们理解它们如何塑造健康和患病细胞功能的一个巨大进步。

Cellular health is determined by the structure, movement, and interplay of its biomacromolecules. Being able to interrogate the behaviour of biomacromolecules within a native cellular context would enable us to gain an enhanced understanding of how these molecules dictate a cells behaviour and function. Proteins are an essential class of biomacromolecule which perform a wide range of cellular processes such as enzyme catalysis, cell signalling and scaffolding, and DNA replication. They consist of a linear chain of amino acids, defined as a polypeptide, their sequence being determined by the genetic sequence which encodes them. An important subset of proteins is integral membrane proteins which reside within cellular membranes and account for about 30% of cellular proteins. Cellular membranes are dynamic structures consisting mostly of protein and lipid which act to compartmentalise the cell, providing barriers to the external environments of the cell and its organelles. Integral membrane proteins are defined by their content of hydrophobic polypeptide stretches which enable parts of their structure to be embedded within, or associated with, the cellular membrane. They are responsible for a variety of dynamic cellular processes, such as sensation, cellular regulation, and cell-to-cell adhesion. A membrane protein's functional capability and their level of expression will largely decide the ionic composition, and therefore the metabolic levels of a given cell type, making them essential for all life, as well as key drug targets. My main aim is to determine structural dynamic information of membrane proteins directly within their native cellular membrane environment, including within live cells. It is important to understand the structural dynamics of proteins, as their fluctuations frequently represent motions and states that are critical for protein function. To do this I will develop general strategies which enable membrane protein structure and dynamics to be deciphered within complex environments by advanced structural mass spectrometry methods. Structural mass spectrometry uses high-resolution mass information on polypeptides and their peptide building blocks to infer on the structural properties of a protein molecule - their shape, interactions, and movements. Using techniques such as hydrogen/deuterium exchange mass spectrometry (which measures the extent and rate of exchange of protein backbone amide hydrogens for deuterium), both global and local information on protein interactions, ligand binding, and structural dynamics can be delivered. Here, I propose the development of chemical biology and advanced mass spectrometry strategies for membrane protein structural investigation within different native membrane environments.One key area in which integral membrane proteins are important is in the development of antimicrobial resistance. Combating antimicrobial resistance is a key societal challenge which, if not addressed, has the potential to become a global health crisis. In bacterial cell lines, the development of multiple drug resistance to structurally unrelated chemicals have been correlated to the function of multidrug efflux membrane protein transporters, which expel a broad range of toxic substances and result in reduced inhibitory effects of antibiotics. My research will focus on developing the aforementioned methods in the context of multidrug efflux membrane protein systems which are known to play major roles in bacterial antibiotic resistance. This will enable an unprecedented insight into the structure, dynamics, and function of these systems, particularly on the impact of drug and lipid interactions, and clinically relevant mutations. More generally, the ability to achieve structural insight into biomacromolecules within cells would be a huge step forward in our understanding of how they shape the function of healthy and diseased cells.